Production of antigen specific donor-derived anti viral T cells for clinical trial use

There are currently four approaches to isolation and expansion of antiviral T-cells for clinical trials: cytokine capture; multimer selection; peptide stimulation; & ex vivo expansion. Due to a relatively large amount of safety and efficacy data associated with the ex-vivo expansion method, primarily out of Baylor College of Medicine, we received FDA approval and have begun producing cytotoxicT cells (CTL) reactive to autologous LCLs that haves been transduced with an adenoviral vector and also express adenovirus and CMV antigens. The methods employed were nearly identical to the methods used at Baylor for generating the CTL shown to be safe and effective against any of the 3 above mentioned viral infections (Leen 2006; Sili 2012). We have produced 5 anti-viral CTL products with this methodology for a trial at Cincinnati Children’s that has a target infusion dose of 50x10⁶ per m² and allows up to 4 monthly infusions at this dose for treatment of post-transplant viral infections. We achieved the target CTL# for 4 infusions with 2 of the 5 cultures. The average number of CTL generated after 3 or 4 stimulations was 398±161 x10⁶ cells with a viability of 79±9%. Including the time required for LCL generation, the CTL took an average of 15 weeks to generate. All cultures remained sterile. The CTL were, on average, 97% CD3⁺, 67% CD4⁺, 31%CD8⁺, and 0.4% CD19⁺. Each CTL culture was tested for its ability to kill transduced LCL and autologous PHA-blasts in a Cr-release assay. Cytotoxicity against transduced LCL averaged 50% ± 10% and averaged 9% ± 7% against autologous blasts at a 25:1 effector: target ratio. No CTL product with >10% cytotoxicity to blasts was released for infusion. It is feasible to produce CTLs with anti-viral activity using this methodology, but the process is lengthy and cumbersome, and the CTL expansion unpredictable. This methodology needs to be improved in order to reliably and reproducibly produce a sufficient # of products for clinical trials.